Abstract

Many conventional chemotherapies act as DNA damaging agents. Chemotherapy activates cell cycle checkpoint signaling to prevent cells from entering mitosis with either damaged or unreplicated DNA. Based on the observations that interference with the checkpoint mechanism (checkpoint override) results in cell death via mitotic catastrophe, the development of checkpoint inhibitors is actively being pursued as an anti-cancer therapy. In a cell-based screen of clinically relevant kinase inhibitors we identified three compounds that display checkpoint override properties: isomer of bosutinib, a recently FDA-approved Abl and Src inhibitor; BEZ235, a PI3K/mTOR inhibitor; and dovitinib, a multi-receptor tyrosine kinase inhibitor. Most interestingly, all compounds display off-target activity towards DNA damage checkpoint kinases including Chk1, ATR and Wee1. We determined these kinase inhibitors potently sensitized a panel of pancreatic cancer cell lines to S-phase acting (Gemcitabine, cisplatin) or G2-phase arresting (doxorubicin) DNA-damaging agents. Using a combination of live-cell videomicroscopy and immunofluorescence, we demonstrated that the inhibitors forced drug-treated cells to override the DNA damage induced checkpoint activation and aberrantly enter mitosis. The aberrant mitoses had characteristic chromatin fragmentation and that kinetochores and centromeres appeared to be detached from the chromatin mass, in a manner reminiscent of mitosis with unreplicated genomes (MUGs). As a result, cells died either in mitosis or due to mitotic catastrophe. The efficacy of pulsatile (24h and 48h after chemotherapy) administration of these previously unsuspected checkpoint inhibitors was further validated in tumor explants and cells isolated directly from a pancreatic cancer patient who is currently undergoing treatment. Tellingly, xenograft studies showed that gemcitabine and bosutinib isomer had significant activity as single agents but the combination significantly prevented tumor growth, even after drug removal. Our findings suggest that kinase inhibitors targeting cell cycle checkpoints, albeit in an off-target manner, may be fast-tracked into the clinic to enhance the efficacy of standard chemotherapies.